Identification and Bioactivities of IFN-γ in Rainbow Trout Oncorhynchus mykiss: The First Th1-Type Cytokine Characterized Functionally in Fish

Abstract

IFN-γ is one of the key cytokines in defining Th1 immune responses. In this study, an IFN-γ homologue has been identified in rainbow trout Oncorhynchus mykiss, and its biological activities have been characterized. The trout IFN-γ cDNA is 1034 bp in length and translates into a 180-aa protein. The first intron of the trout IFN-γ gene contains highly polymorphic GACA minisatellites and 44-bp DNA repeats, giving rise to at least six alleles. IFN-γ is structurally conserved among vertebrates, and a signature motif has been identified. A nuclear localization sequence known to be crucial for IFN-γ biological activities is also present in the C-terminal region of the trout IFN-γ. The IFN-γ expression was induced in head kidney leukocytes by stimulation with PHA or poly(I:C) and in kidney and spleen of fish injected with poly(I:C). rIFN-γ produced in Escherichia coli significantly stimulated gene expression of IFN-γ-inducible protein 10 (γIP-10), MHC class II β-chain, and STAT1, and enhanced respiratory burst activity in macrophages. Deletion of 29-aa residues from the C terminus containing the nuclear localization sequence motif resulted in loss of activity with respect to induction of γIP-10 in RTS-11 cells. Moreover, IFN-γ-induced γIP-10 expression was completely abolished by the protein kinase C inhibitor staurosporine, and partially reduced by U0126, a specific inhibitor for ERKs. Taken together, the present study has demonstrated for the first time a functional IFN-γ homologue in a fish species, strongly suggesting a conserved Th1 immune response is most likely present in lower vertebrates.

Comparative study and expression analysis of the interferon gamma gene locus cytokines in Xenopus tropicalis

“…Previous studies have shown that IL-26 was produced after viral infection or poly(I:C) stimulation (Fickenscher and Pirzer 2004). Elevated transcript level of IFN-γ was observed in several tissues after LPS or poly(I: C) stimulation, in agreement with studies performed in other vertebrates (Schroder et al 2004;Zou et al 2005).…”

“…Apart from conserved structural similarities, the IL-10 cytokine family genes share a five-exon and four-intron genomic organization except for that of the IFN-γ gene which has four exons and three introns. Furthermore, comparative genome analysis has shown that conserved IL-10 and IFN-γ loci are present in the genomes of lower vertebrates such as fish (Zou et al 2003(Zou et al , 2004(Zou et al , 2005Igawa et al 2006) and the gene synteny of the cytokines in these two loci is relatively conserved. Strikingly, the transcription orientation of the cytokine genes and the phase of the introns remain the same during evolution.…”

Cloning of two rainbow trout nucleotide-binding oligomerization domain containing 2 (NOD2) splice variants and functional characterization of the NOD2 effector domains

“…The cells were passaged into fresh culture flasks 2 days before being stimulated for 6 h with either 20 ng/ml recombinant trout IL-1b (rIL-1b) protein [22], or 20 ng/ml recombinant trout interferon-g protein (rIFN-g) [23] or left untreated as control. The RNA preparation and real-time PCR analysis were as described above.…”

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

“…All identified fish IFNs have genomic structures similar to mammalian type III IFN (IFN-) but protein sequences homologous to type I IFN (IFN-␣/␤) (Robertsen, 2006). Type II IFNs (IFN-␥) are most conserved, and they have been described in many fish species (Zou et al, 2005;Igawa et al, 2006). The genes relevant to type I IFN signaling have also been reported, such as melanoma differentiation-associated gene 5 (Mda5), toll-like receptors (TLRs), IFN receptors, IFN regulatory factor (IRF) and several downstream signaling members and ISGs (Robertsen, 2006), demonstrating that teleost fish possess an IFN antiviral system.…”